Heterodimers of glutamic acid

ABSTRACT

Compounds of Formula (Ia) 
                         
wherein R is a C 6 -C 12  substituted or unsubstituted aryl, a C 6 -C 12  substituted or unsubstituted heteroaryl, a C 1 -C 6  substituted or unsubstituted alkyl or —NR′R′,
     Q is C(O), O, NR′, S, S(O) 2 , C(O) 2  (CH2)p   Y is C(O), O, NR′, S, S(O) 2 , C(O) 2  (CH2)p   Z is H or C 1 -C 4  alkyl,   R′ is H, C(O), S(O) 2 , C(O) 2 , a C 6 -C 12  substituted or unsubstituted aryl, a C 6 -C 12  substituted or unsubstituted heteroaryl or a C 1 -C 6  substituted or unsubstituted alkyl, when substituted, aryl, heteroaryl and alkyl are substituted with halogen, C 6 -C 12  heteroaryl, —NR′R′ or COOZ, which have diagnostic and therapeutic properties, such as the treatment and management of prostate cancer and other diseases related to NAALADase inhibition. Radiolabels can be incorporated into the structure through a variety of prosthetic groups attached at the X amino acid side chain via a carbon or hetero atom linkage.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/271,549, now allowed, filed on Oct. 12, 2011; which is a firstdivisional of parent application, U.S. patent application Ser. No.11/936,659 filed on Nov. 7, 2007, now abandoned; which claims priorityto and the benefit of U.S. Provisional Patent Application No. 60/857,490filed Nov. 8, 2006, and U.S. Provisional Patent Application No.60/878,678, filed on Jan. 5, 2007, both now expired; the disclosures ofeach of the foregoing are incorporated herein by reference in theirentireties.

BACKGROUND OF THE INVENTION

At least 1 million men suffer from prostate cancer and it's estimatedthat the disease will strike one in six U.S. men between the ages of 60and 80. There are more than 300,000 new cases of prostate cancerdiagnosed each year. Prostate cancer will affect one in six men in theUnited States, and the mortality from the disease is second only to lungcancer. An estimated $2 billion is currently spent worldwide onsurgical, radiation, drug therapy and minimally invasive treatments, $1billion of the spending in the U.S. There is presently no effectivetherapy for relapsing, metastatic, androgen-independent prostate cancer.New agents that will enable rapid visualization of prostate cancer andspecific targeting to allow radiotherapy present are needed.

N-acetylated alpha-linked acidic dipeptidase (NAALADase), also known asglutamate carboxypeptidase II (GCPII) is a neuropeptidase which cleavesN-acetylaspartyl-glutamate (NAAG) into N-acetylaspartate and glutamatein the nervous system, see below, depicting hydrolytic cleavage of NAAGby NAALDase through the tetrahedral intermediate. The enzyme is a typeII protein of the co-catalytic class of metallopeptidases, containingtwo zinc atoms in the active site.

Independent of its characterization in the nervous system, one form ofNAALADase was shown to be expressed at high levels in human prostaticadenocarcinomas and was designated the prostate-specific membraneantigen (PSMA). The NAALADase/PSMA gene is known to produce multiplemRNA splice forms and based on previous immunohistochemical evidence, ithas been assumed that the human brain and prostate expressed differentisoforms of the enzyme.

Human prostate-specific membrane antigen (PSMA), also known as folatehydrolase I (FOLH1), is a trans-membrane, 750 amino acid type IIglycoprotein which is primarily expressed in normal human prostateepithelium but is upregulated in prostate cancer, including metastaticdisease. PSMA is a unique exopeptidase with reactivity towardpoly-gamma-glutamated folates, capable of sequentially removing thepoly-gamma-glutamyl termini. Since PSMA is expressed by virtually allprostate cancers and its expression is further increased in poorlydifferentiated, metastatic and hormone-refractory carcinomas, it is avery attractive target for prostate imaging and therapy. Developingligands that interact with PSMA and carry appropriate radionuclides mayprovide a promising and novel targeting option for the detection,treatment and management of prostate cancer.

The radio-immunoconjugate form of the anti-PSMA monoclonal antibody(mAb) 7E11, known as the PROSTASCINT scan, is currently being used todiagnose prostate cancer metastasis and recurrence. Early promisingresults from various Phase I and II trials have utilized PSMA as atherapeutic target. PROSTASCINT targets the intracellular domain of PSMAand is thought to bind mostly necrotic portions of prostate tumor.¹⁴More recently, monoclonal antibodies have been developed that bind tothe extracellular domain of PSMA and have been radiolabeled and shown toaccumulate in PSMA-positive prostate tumor models in animals.

While monoclonal antibodies hold promise for tumor detection andtherapy, there have been limited clinical successes outside of lymphomabecause of their low permeability in solid tumors. Low molecular weightmimetics, with higher permeability in solid tumors will have a definiteadvantage in obtaining high percent per gram and a high percentage ofspecific binding.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to compounds of Formula (I)

wherein R is a C₆-C₁₂ substituted or unsubstituted aryl, a C₆-C₁₂substituted or unsubstituted heteroaryl, a C₁-C₆ substituted orunsubstituted alkyl or —NR′R′,

-   Q is C(O), O, NR′, S, S(O)₂, C(O)₂ (CH2)p-   Y is C(O), O, NR′, S, S(O)₂, C(O)₂ (CH2)p-   Z is H or C₁-C₄ alkyl,-   m is 0, 1, 2, 3, 4 or 5-   n is 0, 1, 2, 3, 4, 5 or 6-   p is 0, 1, 2, 3, 4, 5 or 6-   R′ is H, C(O), S(O)₂, C(O)₂, a C₆-C₁₂ substituted or unsubstituted    aryl, a C₆-C₁₂ substituted or unsubstituted heteroaryl or a C₁-C₆    substituted or unsubstituted alkyl, when substituted, aryl,    heteroaryl and alkyl are substituted with halogen, C₆-C₁₂    heteroaryl, —NR′R′ or COOZ    further wherein-   (i) at least one of R or R′ is a C₆-C₁₂ aryl or C₆-C₁₂ heteroaryl    substituted with a halogen or-   (ii) at least one of R or R′ is a C₆-C₁₂ heteroaryl    or a pharmaceutically acceptable salt of the compound of Formula    (I).

Another aspect of the present invention relates to compounds of Formula(Ia)

wherein R is a C₆-C₁₂ substituted or unsubstituted aryl, a C₆-C₁₂substituted or unsubstituted heteroaryl, a C₁-C₆ substituted orunsubstituted alkyl or —NR′R′,

-   Q is C(O), O, NR′, S, S(O)₂, C(O)₂ (CH2)p-   Y is C(O), O, NR′, S, S(O)₂, C(O)₂ (CH2)p-   Z is H or C₁-C₄ alkyl,-   m is 0, 1, 2, 3, 4 or 5-   n 0, 1, 2, 3, 4, 5 or 6-   n′ 0, 1, 2, 3, 4, 5 or 6-   p is 0, 1, 2, 3, 4, 5 or 6-   R′ is H, C(O), S(O)₂, C(O)₂, a C₆-C₁₂ substituted or unsubstituted    aryl, a C₆-C₁₂ substituted or unsubstituted heteroaryl or a C₁-C₆    substituted or unsubstituted alkyl, when substituted, aryl,    heteroaryl and alkyl are substituted with halogen, C₆-C₁₂    heteroaryl, —NR′R′ or COOZ    further wherein-   (i) at least one of R or R′ is a C₆-C₁₂ aryl or C₆-C₁₂ heteroaryl    substituted with at least a halogen or-   (ii) at least one of R or R′ is a substituted or unsubstituted    C₆-C₁₂ heteroaryl    or a pharmaceutically acceptable salt of the compound of Formula    (I).

In a preferred embodiment of the compounds of Formulas (I), (Ia), (II)or (IIa) n is 0 or 1 and n′ is 0 or 1.

The present invention also relates to glutamate-urea-lysine PSMA-bindingmoieties and their use in diagnostic and therapeutic treatment. In oneembodiment, the urea-based analogues described here are glutamate-urea-αor β-amino acid heterodimer coupled through the α-NH₂ or β-NH₂ groups.Radiolabels can be incorporated into the structure through a variety ofprosthetic groups attached at the X amino acid side chain via a carbonor hetero atom linkage. The compounds of the present invention can finduse as targeting agents and diagnostic and therapeutic agents for thetreatment and management of prostate cancer and other diseases relatedto NAALADase inhibition.

Suitable chemical moieties, definitions of chemical moieties, excipientsand methods and modes of administration can be found in US PublishedApplication Nos. 2004/0054190 and 2004/0002478 and InternationalApplication Nos. WO 02/22627 and WO 03/060523, which are incorporated byreference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are HPLC chromatograms respectively of the co-injection ofthe TC-99m-glu-urea-DpK (Tc-99m-MIP 1008), the rhenium analog, and therhenium diester complexes.

FIGS. 2A-2D show stability of the Tc-99m complex of Glu-urea-DpK(Tc-99m-MIP 1008) at 37° C. in respectively PBS pH 7.2, 0.1 M Cysteinein PBS, 0.1 M DTPA in PBS, and 5% mouse serum in PBS for 6 hours.

FIGS. 3A-3B are respective HPLC chromatograms ofN—[N—[(S)-1,3-dicarboxypropyl]carbamoyl]-S-3-iodo-L-tyrosine (I-131DCIT) crude reaction FIG. 3A, top, purified at 2 hours, FIG. 3B, middleand at 2 days FIG. 3C, bottom.

FIG. 4A-4D are radio-HPLC chromatograms of I-131 MIP 1072 purified; at 3days in A) DMSO. B) 3% genstisate-6% Ascorbate/Ascorbic acid, C) PBS,pH=7.2, D) 10% Ethanol in Saline at 37° C. As shown above, theI-131-1072 (peak 12 minutes) remained stable throughout the experiments.

FIG. 5 shows I-123 DCIT bound specifically to LnCap cells and not PC3cells (left set) as is evident by the counts displaceable bynonradiolabeled compound (middle set) or PMPA (right set) in LnCapcells. The histograms show the mean±SEM, each experiment was performedin duplicate.

FIG. 6 is Scatchard Analysis in PSMA Cellular Binding Assay with cold2-{3-[1-Carboxy-2-(4-hydroxy-phenyl)-ethyl]-ureido}-pentanedioic acid(DCT).

FIG. 7 shows biological assessments of selected compounds in thePSMA-positive LNCaP cells.

FIG. 8 show biological assessments of lead compounds in thePSMA-positive LNCaP cells.

FIG. 9 Shows Scatchard Analysis in PSMA Cellular Binding Assay withMIP1072.

FIG. 10 shows internalization of I-131-MIP1072.

FIGS. 11A and 11B respectively show stability of I-131 MIP-1072 verses,DCT and Phenacetin in rat microsomes for 60 minutes.

FIG. 12 shows tissue biodistribution of the I-131 MIP1072 in xenografttumored mice.

FIG. 13 shows inhibition of NAALADase activity from LNCaP cellularlysates.

FIG. 14 shows inhibition of NAALADase Activity from LNCaP Cellularlysates.

FIG. 15 shows inhibition of NAALADase Activity from LNCaP Cellularlysates.

FIG. 16 shows the ability of test compounds to inhibit the binding of aknown NAALADase inhibitor, 131I-DCIT, to PSMA on LNCaP cells wasexamined. Cells were incubated with various concentrations of testcompounds and 131I-DCIT for 1 hour then washed and counted to determineIC50 values.

FIG. 17 is direct binding analysis of MIP-1072. ¹²³I-MIP-1072 (3nM, >1,000 mCi/μmole) was incubated with PSMA positive LNCaP cells orPSMA negative PC3 cells (300,000 cells/well), in both the absence andpresence of either non-radioactive 10 μM MIP-1072 or 10 μM of a specificPSMA inhibitor (PMPA).

FIG. 18 shows saturation binding analysis of ¹²³I-MIP-1072 in LNCaPcells.

FIG. 19 shows internalization of ¹²³I-MIP-1072.

FIG. 20 shows uptake of ¹²³I-MIP-1072 in LNCaP xenograft bearing mice.Tissue biodistribution of ¹²³I-MIP-1072 (2 μCi/mouse, >1,000 mCi/μmole)was assessed in selected tissues from LNCaP (PSMA positive) tumorbearing nude mice. Results are expressed as the percent of the injecteddose per gram of the selected tissues (% ID/g).

FIG. 21 Uptake of ¹²³I-MIP-1072 in LNCaP and PC3 xenograft bearing mice.Tissue biodistribution of ¹²³I-MIP-1072 (2 μCi/mouse, >1,000 mCi/μmole)was assessed in selected tissues from LNCaP (PSMA positive) and PC3(PSMA negative) tumor bearing nude mice with (Blocked) or without(Normal) pretreatment with 50 mg/kg PMPA.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

-   “Pharmaceutically acceptable salt” refers to those salts which    retain the biological, effectiveness and properties of the free    bases and which are obtained by reaction with inorganic acids such    as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,    phosphoric acid, methanesulfonic acid, ethanesulfonic acid,    p-toluenesulfonic acid, salicylic acid and the like.-   “Alkyl” refers to a straight-chain, branched or cyclic saturated    aliphatic hydrocarbon. Typical alkyl groups include methyl, ethyl,    propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl    and the like. The alkyl group may be optionally substituted with one    or more substituents selected from the group consisting of hydroxyl,    cyano, or alkoxy. When the alky group is an R′ substituent, it is a    lower alkyl of from 1 to 6 carbons, more preferably 1 to 4 carbons.-   “Aryl” refers to an aromatic group which has at least one ring    having a conjugated pi electron system and includes carbocyclic    aryl, heterocyclic aryl and biaryl groups. The aryl group may be    optionally substituted with one or more substituents selected from    the group consisting of halogen, trihalomethyl, hydroxyl, SH, OH,    NO₂, amine, thioether, cyano, alkoxy, alkyl, and amino. Examples of    aryl groups include phenyl, napthyl and anthracyl groups. Phenyl and    substituted phenyl groups are preferred.-   “Heteroaryl” refers to an aryl group having from 1 to 3 heteroatoms    as ring atoms, the remainder of the ring atoms being carbon.    Heteroatoms include oxygen, sulfur, and nitrogen. Thus, heterocyclic    aryl groups include furanyl, thienyl, pyridyl, pyrrolyl, N-lower    alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like.    Synthesis

All reactions were carried out in dry glassware under an atmosphere ofargon unless otherwise noted. Reactions were purified by columnchromatography, under medium pressure using a Biotage SP4 or bypreparative high pressure liquid chromatography.

¹H NMR was recorded on a Bruker 400 MHz instrument. Spectra are reportedas ppm δ and are referenced to the solvent resonances in CDCl₃, DMSO-d₆or methanol-d₄. All solvents were purchased from Sigma-Aldrich. Reagentswere purchased from Sigma Aldrich, Bachem, Akaal, Fisher, Alfa Aesar,Acros and Anaspec. The following abbreviations are used methylenechloride (DCM), ethyl acetate (EA), hexanes (Hex), dichloroethane (DCE),dimethyl formamide (DMF), trifluoroacetic acid (TFA), tetrahydrofuran(THF), carbonyldiimidazole (CDI), dimethylaminopyridine (DMAP),triethylamine (TEA), methyl trifluoromethanesulfonate (MeOTf),(S)-2-Amino-6-(bis-pyridin-2-ylmethyl-amino)-hexanoic acid (dpK),glutamic acid (Glu), diisopropylethylamine (DIEA), benzyloxycarbonyl(CBZ).

Synthesis of Intermediates

The following compounds were all prepared in overall yields ranging from20-40% following the route depicted in Scheme 1. The first step,performed at 0° C. under inert conditions used the di-t-butyl ester ofGlutamic acid with CDI in the presence of base to form the intermediateGlu-urea-imidazole derivative 2. This intermediate was activated withMeOTf under basic conditions to afford the methylated imidazole 3, whichunder inert conditions reacted readily with amines. The tert-butyl esterprotecting groups were removed using 20% TFA in DCM for 1 to 4 hour atroom temperature. Upon completion of the deprotection, the reactionswere concentrated on a rotary evaporator or blown dry with nitrogen andpurified on a silica column or recrystallized. The final products weretested in vitro and in vivo.

L-(S)-2-[(Imidazole-1-carbonyl)-amino]-pentanedioic acid di-tert-butylester (2)

To a suspension of di-t-butyl glutamate hydrochloride (15.0 g, 51 mmol)in DCM (150 mL) cooled to 0° C. was added TEA (18 mL) and DMAP (250 mg).After stirring for 5 min. CDI (9.0 g, 56 mmol) was added and thereaction was stirred overnight with warming to room temperature. Thereaction was diluted with DCM (150 mL) and washed with saturated sodiumbicarbonate (60 mL), water (2×100 mL) and brine (100 mL). The organiclayer was dried over sodium sulfate and concentrated to afford the crudeproduct as a semi-solid, which slowly solidified upon standing. Thecrude material was triturated with hexane/ethyl acetate to afford awhite solid which was filtered, washed with hexane (100 mL) and dried toafford the desired product (15.9 g, 45 mmol, 88%) as a white solid. ¹HNMR (400 MHz, DMSO-d₆) δ 7.63 (s, 1H), 7.00 (br, 2H), 6.31 (d, 1H), 4.02(m, 1H), 2.19 (m, 2H), 1.86 (m, 1H), 1.67 (m, 1H), 1.39 (s, 9H), 1.38(s, 9H). ESMS m/z: 354 (M+H)⁺.

Alternatively, the analogs can be prepared via the isocyanate generatedin situ using triphosgene. This approach can be accomplished by eitheractivation of the glutamate residue and coupling with a lysine residue(route A) or by activating the lysine residue and coupling it with theglutamate (route B) as shown in scheme 2 below.

L-(S,S)-2-[3-(5-Benzyloxycarbonylamino-1-tert-butoxycarbonyl-pentyl-ureido)-pentanedioicacid di-tert-butyl ester (3)

-   Route A. In a round bottom flask 1.8 mL TEA (13.2 mmol) was combined    with 1.8 grams (6 mmol) L-glutamic acid di-tertbutyl ester    hydrochloride in 20 mL DCM. This solution is added dropwise over 45    minutes to a solution of 10 mL DCM and triphosgene (0.7 g, 2.2 mmol)    at 0° C. After stirring an additional 30 min a solution of    H-lys-(Z)—O-t-butyl ester HCl (2.2 g, 6 mmol) containing TEA (1.8    mL, 13 mmol) in 15 mL DCM was added in one portion. The solution was    stirred for 1 hour. The reaction is concentrated, diluted with 50 mL    ethyl acetate, washed 2N NaHSO4 (2×50 mL), brine (50 mL) and dried    over sodium sulfate to yield a yellow oil. Purification by column    chromatography to afford the desired product as a clear oil which    upon standing solidifies to a white solid (1.9 g, 54%).-   Route B. In a round bottom flask triphosgene (2.9 g, 10 mmol) is    suspended in DCM (50 mL) and stirred at 0° C. A solution of    H-Lysine(Z) freebase (9.1 g, 27 mmol) and DIEA (10.4 mL, 60 mmol)    DCM (50 mL) was added dropwise to the triphosgene solution over 2.5    hours. After 2.5 hours a solution of L-glutamic acid di-tertbutyl    ester hydrochloride (8 g, 27 mmol) containing DIEA (10.4 mL, 60    mmol) DCM (50 mL) was added in one portion and allowed to stir for    45 minutes. The reaction was concentrated to dryness, diluted with    150 mL ethyl acetate, washed with 2N NaHSO₄ (2×200 mL), brine (150    mL) and dried over sodium sulfate to yield a yellow oil. This oil    was purified by column chromatography (SiO₂) to afford the desired    product as a clear oil which upon standing solidifies to a white    solid (12.0 g, 72%). ¹H NMR (400 MHz, CDCl₃) δ 7.34 (m, 5H),    5.33-5.28 (m, 3H), 5.08 (d, J=7.4 Hz, 2H), 4.38-4.29 (m, 2H), 3.15    (m, 2H), 2.32-2.01 (m, 2H), 1.90-1.50 (m, 8H), 1.43-1.40 (m, 27H,    t-Bu's). ESMS m/z: 622 (M+H)⁺.

2-[3-(5-Amino-1-tert-butoxycarbonyl-pentyl)-ureido]-pentanedioic aciddi-tert-butyl ester (4)

To a solution of2-[3-(5-Benzyloxycarbonylamino-1-tert-butoxycarbonyl-pentyl)-ureido]-pentanedioicacid di-tert-butyl ester (630 mg, 1.0 mmol) in ethanol (20 mL) was addedammonium formate (630 mg, 10 eqv) followed by 10% Pd—C and thesuspension was allowed to stand with occasional agitation overnightuntil complete. The reaction was filtered through celite andconcentrated to afford the desired product (479 mg, 98%) as a waxysolid. ¹H NMR (400 MHz, CDCl₃) δ 7.15-6.0 (bm, 4H, NH's), 4.29 (m, 2H),3.02 (m, 2H), 2.33 (m, 2H), 2.06-1.47 (m, 8H), 1.45-1.40 (m, 27H,t-Bu's). ESMS m/z: 488 (M+H)⁺.

Synthesis of the Glu-Urea-Glu Tether Core Model Compounds

In this series a tether is incorporated onto the side chain of glutamicacid or lysine prior to conjugation to form the urea dimer. In theexample below the side chain carboxylic acid of one of the glutamicacids is modified into a tether to append a chelator, atom or functionalgroup that is or contains a radionuclide (Scheme 4).

2-{3-[3-(4-Amino-butylcarbamoyl)-1-methoxycarbonyl-propyl]-ureido}-pentanedioicacid di-tert-butyl ester (28)

To a solution of N-BOC Glutamic acid α-methyl ester BOC-Glu(OH)-Ome (960mg, 3.7 mmol) in DMF (6 mL) cooled to 0° C. was added EDC (845 mg, 1.3eqv) and TEA (1.3 mL). After stirring for 10 min the mono protecteddiamine N-CBZ-1,4-diaminobutane hydrochloride salt (1 g, 3.8 mmol) wasadded and the reaction is allowed to stir overnight with warming to roomtemperature. The crude reaction was diluted with EA (100 mL) and washedwith and washed with water (30 mL), 5% aq. Citric acid (30 mL), sat.sodium bicarbonate (30 mL), water (30 mL) and brine (30 mL). The organiclayer was dried over sodium sulfate and concentrated to afford the crudeproduct as a thick syrup (2.1 g). To the obtained syrup was added 4 NHCl in dioxane (10 mL) and the reaction was stirred at room temperaturefor 3 h. Concentration afforded a waxy solid (1.8 g) as thehydrochloride salt. The salt was coupled to the activatedL-(S)-2-[(Imidazole-1-carbonyl)-amino]-pentanedioic acid di-tert-butylester (2) as described in the preceding experimental sections to affordthe desired fully protected dimer×(1.9 g). This material was suspendedin absolute EtOH (20 mL) excess ammonium formate (5 g) added followed by20% Pd(OH)₂ on carbon (100 mg) and the suspension very gently agitatedovernight to effect cleavage of the CBZ protection group. Filtrationthrough celite and concentration afforded the desired free amine (1.4 g,2.7 mmol, 73%, 4 steps). ¹H NMR (400 MHz, CDCl₃) δ 8.41 (br, 2H), 7.36(br, 1H), 6.44 (bs, 1H), 6.37 (bs, 1H), 4.37-4.29 (m, 2H), 3.71 (s, 3H),3.20-1.50 (m, 16H), 1.45 (s, 9H), 1.43 (s, 9H). ESMS m/z: 517 (M+H)⁺.

Re(CO)₃-2-(3-{3-[4-(Bis-pyridin-2-ylmethyl-amino)-butylcarbamoyl]-1-carboxy-propyl}-ureido)-pentanedioicacid[Br] (29) (MIP-1100)

The protected intermediate was prepared by reductive amination usingpyridine-2-carboxaldehyde as previously described. Treatment with 2MLiOH in MeOH effected hydrolysis of the methyl ester. The methanol wasremoved and excess DCM:TFA (1:1) was added and the reaction stirred atroom temperature overnight. The crude material was converted into thedesired Rhenium conjugate following the procedure described above.Preparative HPLC afforded the desired molecule (9.5 mg, 16%). ¹H NMR(400 MHz, DMSO-d₆) δ 8.78 (m, 2H), 8.31 (br, 1H), 7.95 (m, 2H), 7.59 (m,2H), 7.39 (m, 2H), 6.60-6.33 (m, 21H), 4.89 (m, 4H), 4.00 (m, 1H), 3.76(m, 1H), 3.20-1.2 (m, 16H) (3 CO₂H not seen). ESMS 842 (M−H)⁺.

Synthesis of the Glu-urea-X-benzyl-Lysine Core Model Compounds

The following compounds were all prepared in overall yields ranging from20-40% using the route depicted in Scheme 3. The Z-deprotectedGlu-urea-lysine was mixed with the appropriate aldehyde (0.9equivalents) at room temperature for one hour to form the □chiff baseintermediate. The □chiff base was reduced using 1 equivalent of sodiumtriacetoxyborohydride. The compounds were deprotected using 50% TFA inDCM for 1 hour at room temperature. Upon completion, the reactions wereconcentrated on a rotary evaporator or were blown dry with nitrogen andextracted using methylene chloride and water. The water layer wasevaporated to dryness to afford the deprotected product in 40-80% yield.

4-Trimethylstannanyl-benzaldehyde (5)

To a solution of 4-iodobenzaldehyde (1.92 g; 8.27 mmol) in dry dioxane(60 mL) was added hexamethylditin (4.1 mL, 19.8 mmol) followed byPd(Ph₃P)Cl₂ (150 mg) and the reaction mixture was heated for 3 h tinderreflux until judged complete. The reaction was filtered through celiteand purified by column chromatography using hexanes/ethyl acetate (9/1)as eluent to afford (2.24 g, 98%) as a clear oil. ¹H NMR (400 MHz,CDCl₃) δ 9.97 (s, 1H) 7.81 (d, J=7.8 Hz, 2H), 7.72 (d, J=7.8 Hz, 2H),0.29 (s, 91). ESMS m/z: 268 (Sn-cluster).

2-{3-[1-tert-Butoxycarbonyl-5-(4-trimethylstannanyl-benzylamino)-pentyl]-ureido}-pentanedioicacid di-tert-butyl ester (6)

To a solution of2-[3-(5-Amino-1-tert-butoxycarbonyl-pentyl)-ureido]-pentanedioic aciddi-tert-butyl ester (150 mg, 0.31 mmol) in DCE (10 mL) was added4-Trimethylstannanyl-benzaldehyde (82 mg, 0.31 mmol) followed by sodiumtriacetoxyborohydride (98 mg, 0.47 mmol) and the reaction was stirredovernight at 40° C. The reaction was concentrated and purified by columnchromatography using hexanes/ethyl acetate as eluent to afford thedesired product (88 mg, 38%) as a thick syrup which begins to solidifyupon standing. ¹H NMR (400 MHz, DMSO-d) δ 7.48 (d, J=7.4 Hz, 2H), 7.30(d, J=7.4 Hz, 2H), 6.27 (m, 2H, NH's), 3.96 (m, 4H), 2.74 (bm, 2H), 2.21(m, 2H), 1.87 (m, 2H), 1.65-1.19 (m, 7H), 1.35 (m, 27H, t-Bu's), 0.23(s, 9H). ESMS n/z: 742 (Sn-cluster).

(S, S)-2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid (7)(MIP 1033)

The same experimental procedure as depicted in scheme 1, yielded 8% of2-[3-(5-benzyloxycarbonylamino-1-tert-butoxycarbonyl-pentyl)-ureido]-pentanedioicacid di-tert-butyl ester. The compound was deprotected using thepreviously described methods and purified by HPLC to afford the desiredproduct. ¹H NMR (tri-t-butyl ester of Z-protected amine) (400 MHz,CDCl₃) δ 12.2 (s, 3H), 6.4 (s, 2H), 4.15 (m, 2H), 3.45 (m, 1H), 2.75(bs, 1H), 2.2 (m, 4H), 1.90 (m, 2H), 1.65 (m, 2H), 1.50 (s, 2H), 1.35(m, 2H). ESMS m/z: 622 (M−H)⁺.

(S)-2-(3,3-Bis-pyridin-2-ylmethyl-ureido)-pentanedioic acid (8) (MIP1025)

The same experimental procedure as in the general synthesis, yielded0.65 g, 48% of 2-(3,3-Bis-pyridin-2-ylmethyl-ureido)-pentanedioic aciddi-tert-butyl ester. The compound was deprotected using the previouslydescribed methods and purified by HPLC to afford the desired product. ¹HNMR (400 MHz, DMSO-d₆) δ, 12.0 (bs, 2H), 8.68 (d, 2H), 8.00 (m, 2H),7.41 (d, 4H), 7.14 (d, 1H), 4.73 (d, 4H), 3.96 (s, 1H), 2.18 (m, 2H),1.80 (m, 2H).

(S,S)-2-{3-[3-(Bis-pyridin-2-ylmethyl-amino)-1-carboxy-propyl]-ureido}-pentanedioicacid (9) (MIP 1028)

The same experimental procedure as in the general synthesis in scheme 1,yielded 0.16 g, 35% of2-{3-[3-(Bis-pyridin-2-ylmethyl-amino)-1-carboxy-propyl]-ureido}-pentanedioicacid di-tert-butyl ester. The compound was deprotected using thepreviously described methods and purified by HPLC to afford the desiredproduct. ¹H NMR (400 MHz, DMSO-d₆) δ 12.4 (br, 2H), 9.37 (s, 1H), 8.52(d, 2H), 7.80 (t, 2H), 7.14 (dd, 4H), 6.45 (m, 2H), 4.49 (br, 4H), 4.12(s, 1H), 4.05 (s, 1H), 3.21 (m, 2H), 2.24 (m, 2H), 1.80 (m, 2H), 1.40(m, 2H). ESMS m/z: (diethyl ester) 429 (M)⁺, 451 (M+Na).

(S,S)-2-{3-[5-(Bis-pyridin-2-ylmethyl-amino)-1-carboxy-pentyl]-ureido}-pentanedioicacid (10) (MIP 1008)

The same experimental procedure as in the general synthesis, yielded0.09 g, 12% of2-{3-[5-(Bis-pyridin-2-ylmethyl-amino)-1-carboxy-pentyl]-ureido}-pentanedioicacid di-tert-butyl ester. The compound was deprotected using thepreviously described methods and purified by HPLC to afford the desiredproduct. ¹H NMR (400 MHz, DMSO-d₆) δ 12.7 (s, 2H), 8.97 (s, 1H), 8.65(dd, 2H), 7.91 (dd, 2H), 7.45 (m, 4H), 6.44 (d, 1H), 6.28 (d, 1H), 4.45(br, 4H), 4.10 (m, 2H), 3.15 (br, 2H), 2.60 (m, 2H), 2.25 (m, 2H), 1.90(m, 2H), 1.78 (m, 2H), 1.45 (m, 2H).

(S)-2-{3-[1-Carboxy-2-(4-iodo-phenyl)-ethyl]-ureido}-pentanedioic acid(11) (MIP-1034)

The same experimental procedure as in the general synthesis, yielded0.038 g, 5% of2-{3-[1-Carboxy-2-(4-iodo-phenyl)-ethyl]-ureido}-pentanedioic aciddi-tert-butyl ester. The compound was deprotected using the previouslydescribed methods. ¹H NMR (400 MHz, DMSO-d₆) δ 12.40 (s, 3H), 7.65 (dd,2H), 7.05 (dd, 2H), 6.30 (m, 2H), 4.25 (s, 1H), 4.05 (s, 1H), 2.90 (m,2H), 2.2 (m, 2H), 1.80 (m, 2H). ESMS m/z: 429 (M)⁺, 451 (M+Na).

(S,S)-2-{3-[1-Carboxy-5-(2-iodo-benzylamino)-pentyl]-ureido}-pentanedioicacid (12) (MIP 1035)

The same general procedure, using the previously prepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester. The compound was deprotected using the previously describedmethods (5.5 mg, 66%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.4 (s, 3H), 8.8 (s,1H), 7.94 (m, 1H), 7.5 (m, 1H), 7.16 (t, 1H), 6.38 (m, 2H), 4.15 (m,5H), 3.06 (s, 2H), 2.85 (s, 1H), 2.2 (m, 2H), 1.90 (m, 1H), 1.70 (m,2H), 1.50 (s, 2H), 1.35 (m, 2H). ESMS m/z: 536 (M+H)⁺.

(S,S)-2-{3-[1-Carboxy-5-(3-iodo-benzylamino)-pentyl]-ureido}-pentanedioic(13) (MIP 1089)

The same general procedure, using previously prepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di-t-butylester. The compound was deprotected using the previously describedmethods (4.1 mg, 53%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.4 (s, 3H), 8.7 (s,2H), 7.9 (s, 1H), 7.8 (d, 1H), 7.44 (d, 1H), 7.22 (t, 1H), 6.25 (s, 2H),4.09 (m, 5H), 2.89 (s, 1H), 2.75 (s, 1H), 2.2 (d, 2H), 1.90 (m, 2H),1.65 (m, 2H), 1.40 (m, 2H).

(S,S)-2-{3-[1-Carboxy-5-(4-iodo-benzylamino)-pentyl]-ureido}-pentanedioic(14) (MIP 1072)

The same general procedure, using previously prepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester. The compound was deprotected using the previously describedmethods (12 mg, 66%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.4 (bs, 3H), 8.8(br, 1H), 7.8 (d, 2H), 7.27 (d, 2H), 6.35 (br, 2H), 4.1 (m, 4H), 2.89(m, 2H), 2.2 (d, 2H), 1.90 (m, 2H), 1.65 (m, 4H), 1.35 (m, 2H). ESMSm/z: 536 (M+H)⁺.

(S,S)-2-{3-[1-Carboxy-5-(4-fluoro-benzylamino)-pentyl]-ureido}-pentanedioic(15) (MIP 1090)

The same general procedure, using previously prepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester. The compound was deprotected using the previously describedmethods. ¹H NMR (400 MHz, DMSO-d₆) δ 12.4 (br, 3H), 8.7 (br, 1H), 7.5(m, 2H), 7.3 (m, 2H), 6.35 (m, 2H), 4.1 (m, 4H), 2.9 (m, 2H), 2.2 (d,2H), 1.90 (m, 2H), 1.60 (m, 4H), 1.35 (m, 2H), ESMS m/z: 428 (M+H)⁺, 450(M+Na).

(S,S)-2-{3-[1-Carboxy-5-(4-bromo-benzylamino)-pentyl]-ureido}-pentanedioic(16) (MIP 1094)

The same general procedure, using previously prepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester. ¹HNMR (tri t-butyl ester) (400 MHz, CDCl₃) δ 7.52 (d, 2H), 7.32(d, 2H), 6.28 (m, 2H), 3.98 (m, 2H), 2.55 (t, 2H), 2.48 (t, 2H), 2.22(m, 2H), 1.85 (m, 2H), 1.62 (m, 2H), 1.45 (m, 2H), 1.37 (s, 27H), 1.28(m, 2H) ESMS m/z: 642 (M+H)⁺. The compound was deprotected using thepreviously described methods. ESMS m/z: 474 (M+H)⁺.

(S,S)-2-{3-[1-Carboxy-5-(4-iodo-benzoylamino)-pentyl]-ureido}-pentanedioicacid (17) (MIP 1044)

The same general procedure, using previously prepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester. The compound was deprotected using the previously describedmethods. ¹H NMR (400 MHz, DMSO-d₆) δ 12.4 (s, 3H), 8.45 (s, 1H), 7.8(dd, 2H), 7.6 (dd, 2H), 6.3 (s, 2H), 5.75 (s, 1H), 4.1 (m, 4H), 3.2 (s,2H), 2.25 (d, 2H), 1.90 (m, 1H), 1.65 (m, 2H), 1.4 (m, 2H).

2-{3-[1-carboxy-5-(4-iodo-benzenesulfonylamino)-pentyl]-ureido}-pentanedioicacid (18). (MIP 1097)

In a round bottom flask2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester (300 mg, 0.62 mmol) is suspended in water (10 mL) and 1,4 dioxane(10 mL) and TEA (1.75 mL, 1.25 mmol) was added followed by4-iodo-benzenesulfonyl chloride and the mixture stirred overnight at 50°C. The reaction mixture was evaporated to dryness, taken up in DCM andchromatographed over silica gel to afford the desired product (375 mg,80%) as a clear oil. The compound was deprotected using the previouslydescribed methods followed by HPLC purification to afford the desiredproduct MIP-1097 as a whiter solid (270 grams, 90% yield). ¹H NMR (400MHz, DMSO-d₆) δ 7.97 (d, 2H), 7.68 (t, 1H), 7.53 (d, 2H), 6.35 (dd, 2H),4.10 (m, 1H), 4.00 (m, 1H), 2.65 (m, 2H), 2.22 (m, 2H), 1.9 (m, 1H), 1.7(m, 1H), 1.55 (m, 1H), 1.45 (m, 1H), 1.35 (m, 2H), 1.25 (m, 2H), (3 CO₂Hnot seen). ESMS m/z: 565 (M+H)⁺.

2-(3-{1-Carboxy-5-[3-(4-iodo-phenyl)-ureido]-pentyl}-ureido)-pentanedioicacid (19) (MIP 1095)

In a round bottom flask 4-iodo-phenyl isocyanate (100 mg, 0.41 mmol) isdissolved in DCM (10 mL) containing TEA (0.057 mL, 0.4 mmol).2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester (200 mg, 0.41 mmol) was added and stirred for 3 hours. Thereaction mixture was evaporated to dryness and the crude mixture takenup in methanol (5 mL). Dropwise addition to water (20 mL) afforded awhite precipitate which was collected and washed with water (20 mL) anddried to afford the desired tri-tert butyl ester as a white solid whichwas deprotected directly using the previously described method to affordthe desired product (158 mg, 53%) as a white solid. ¹H NMR (400 MHz,DMSO-d₆) δ 8.51 (s, 1H), 7.5 (d, 2H), 7.22 (d, 2H), 6.3 (t, 2H), 6.16(t, 1H), 4.05 (m, 2H), 3.05 (m, 2H), 2.24 (m, 2H), 1.9 (m, 1H), 1.68 (m,2H), 1.52 (m, 1H), 1.38 (m, 2H), 1.28 (m, 2H), (3 CO₂H not seen). ESMSm/z: 565 (M+H)⁺.

Synthesis of Glu-Urea-β-Phenyl Glycines

(±)3-Amino-3-(3-iodo-phenyl)-propionic acid (20)

Malonic acid (2.2 g, 21.5 mmol) and 3-iodobenzaldehyde (5 g, 21.5 mmol)were suspended in ethanol (50 mL) and ammonium acetate (1.66 g, 21.5mmol) was added and the reaction heatred to a reflux overnight. Thereaction was cooled to room temperature filtered and washed with ethanolfollowed by ether and dried to afford the product (3.4 g, 11.6 mmol,54%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 7.80 (s, 1H), 7.64(dd, J=7.8 Hz, 1H), 7.42 (dd, J=7.6 Hz, 1H), 7.16 (dd, J=7.8 Hz, 1H),7.14 (dd, J=7.6 Hz, 1), 4.21 (m, 1H), 2.36 (m, 2H).

(±)-3-Amino-3-(3-iodo-phenyl)-propionic acid methyl ester (21)

To a suspension of (±)3-Amino-3-(3-iodo-phenyl)-propionic acid (3.1 g,10.6 mmol) in methanol was added thionyl chloride (0.95 mL, 12.7 mmol)and the reaction was stirred at room temperature overnight.Concentration followed by trituration with ether gives a white solid.The solid is filtered, washed with ether and dried to afford the desiredproduct (3.5 g, 10 mmol, 95%) as a white solid. ¹H NMR (400 MHz,DMSO-d₆) δ 8.79 (br, 2H), 8.01 (s, 1H), 7.74 (d, J=8.1 Hz, 1H), 7.57 (d,J=7.8 Hz, 1H), 7.21 (dd, J=8.1, 7.8 Hz, 1H), 4.56 (br, 1H), 3.54 (s,3H), 3.23-3.17 (m, 1H), 3.04-2.98 (m, 1H).

(S, R) and (S,S)-2-{3-[1-(3-lodo-phenyl)-2-methoxycarbonyl-ethyl]-ureido}-pentanedioicacid di-tert-butyl ester (22)

2-[(Imidazole-1-carbonyl)-amino]-pentanedioic acid di-tert-butyl ester(370 mg, 1.05 mmol) was dissolved in DCE (10 mL) and coiled to 0° C.MeoTf (142 μL, 1.25 mmol) was added and the reaction was allowed toproceed for 20 min. (±)3-Amino-3-(3-iodo-phenyl)-propionic acid methylester (356 mg, 1.045 mmol) was added and the reaction was allowed towarm to room temperature and then warmed to 55° C. and stirredovernight. The reaction was diluted with DCM (50 mL) and washed withwater (30 mL), 5% aq. Citric acid (30 mL), sat. sodium bicarbonate (30mL), water (30 mL) and brine (30 mL). The organic layer was dried oversodium sulfate and concentrated to afford the crude product. The productwas puridied by column chromatography to afford the desired product (303mg, 0.51 mmol, 49%) as a white foam. ¹H NMR (400 MHz, CDCl₃) δ 7.66 (s,1H), 7.57 (d, J=7.6 Hz, 1H), 7.29 (s, 1H), 7.07-7.02 (m, 1H), 5.74 (br,1H), 5.17 (br, 2H), 4.30 (m, 1H), 3.63 (s, 1.5H), 3.62 (s 1.5H),2.88-2.76 (m, 2H), 2.38-2.24 (m, 2H), 2.10-2.00 (m, 1H), 1.90-1.80 (m,1H), 1.46 (s, 9H), 1.44 (s, 9H).

(S, R) and (S,S)-2-{3-[2-Carboxy-1-(3-iodo-phenyl)-ethyl]-ureido}-pentanedioic acid(23)

To a solution of(±)2-{3-[1-(3-lodo-phenyl)-2-methoxycarbonyl-ethyl]-ureido}-pentanedioicacid di-tert-butyl ester (289 mg, 0.49 mmol) was dissolved in methanol(3 mL) and 2M LiOH (0.5 mL) was added and the reqaction stirred at roomtemperature overnight. The reaction was diluted with water (20 mL) andthe organic layer was extracted with ethyl acetate (2×20 mL) thenacidified with 1N HCl to pH ˜2. The aqueous layer was extracted withethyl acetate (3×20 mL), dried over sodium sulfate and concentrated toafford the crude product (206 mg, 0.36 mmol, 73%) as a white solid. Tothe crude material was added DCM (2 mL) followed by TFA (2 mL) and thereaction was stirred at room temperature overnight. Concentrationfollowed by recrystallization from ethyl acetate afforded the desiredproduct (22 mg, 0.047 mmol, 10%) as a white solid. ¹H NMR (400 MHz,DMSO-d₆) δ 12.39 (br, 3H), 7.64 (br, 1H), 7.56 (m, 1H), 7.30 (bm, 1H),7.10 (bm, 1H), 6.72 (bm, 1H), 6.34 (bm, 1H), 4.94 (br, 1H), 4.03 (bm,1H), 2.64 (br, 2H), 2.20 (br, 2H), 1.86 (br, 1H), 1.71 (br, 1). ESMSm/z: 463 (M−H)⁺.

(S,S)-2-{3-[1-Carboxy-5-(2-chloro-benzylamino)-pentyl]-ureido}-pentanedioic(7) (MIP-1137)

The same general procedure as shown in Scheme 1, using previouslyprepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di-t-butylester. The compound was deprotected using the previously describedmethods to yield the desired product (100 mg, 45%) as an off-whitesolid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.0 (br, 3H), 7.63 (d, 1H), 7.2 (m,2H), 7.15 (d, 1H), 6.30 (d, 2H), 4.1 (m, 4H), 2.9 (br, 2H), 2.2 (m, 2H),1.90 (m, 2H), 1.60 (m, 4H), 1.35 (m, 2H). ESMS m/z: 444 (M+H)⁺.

(S,S)-2-{3-[1-Carboxy-5-(3-chloro-benzylamino)-pentyl]-ureido}-pentanedioic(8) (MIP 1131)

The same general procedure as shown in Scheme 1, using previouslyprepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester. The compound was deprotected using the previously describedmethods to yield the desired product (200 mg, 90%) as an off-whitesolid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.9 (br, 3H), 7.6 (s, H), 7.43 (m,3H), 6.39 (br, 2H), 4.1 (m, 4H), 2.9 (br, 2H), 2.2 (m, 2H), 1.90 (m,2H), 1.60 (m, 4H), 1.35 (m, 2H). ESMS m/z: 444 (M+H)⁺.

(S,S)-2-{3-[1-Carboxy-5-(4-chloro-benzylamino)-pentyl]-ureido}-pentanedioic(9) (MIP 1135)

The same general procedure as shown in Scheme 1, using previouslyprepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester. The compound was deprotected using the previously describedmethods to yield the desired product as (10 mg, 66%) as an off-whitesolid. ESMS m/z: 444 (M+H)⁺.

(S)-2-(3-((R)-5-(benzylamino)-1-carboxypentyl)ureido)pentanedioic acid(10). (MIP-1106)

The same general procedure as shown in Scheme 1, using previouslyprepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester. The compound was deprotected using the previously describedmethods to yield the desired product (5 mg, 47%) as an off-white solid.ESMS m/z: 410 (M+H)⁺.

2-(3-{1-Carboxy-5-[3-(phenyl)-ureido]-pentyl}-ureido)-pentanedioic acid(11) (MIP 1111)

In a round bottom flask phenyl isocyanate (100 mg, 0.84 mmol) wasdissolved in DCM (10 mL)2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester (409 mg, 0.84 mmol) was added and stirred for 3 hours. Thereaction mixture was evaporated to dryness and the crude mixture waspurified via flash column chromatography 2:1 hexanes/ethyl acetate toafford the tert-butyl ester as a white solid which was deprotected usingTFA/CH₂Cl₂ affording the desired product. ¹H NMR (400 MHz, DMSO-d₆) δ12.5 (s, 3H), 8.54 (s, 1H), 7.40 (dd, 2H), 7.26 (dd, 2H), 6.30 (t, 2H),6.17 (t, 1H), 4.05 (m, 2H), 3.05 (m, 2H), 2.44 (m, 2H), 1.90 (m, 1H),1.68 (m, 2H) 1.52 (m, 1H), 1.40 (m, 2H), 1.29 (m, 2H). ESMS m/z: 439(M+H)⁺.

2-(3-{1-Carboxy-5-[3-(4-bromo-phenyl)-ureido]-pentyl}-ureido)-pentanedioic-acid(12) (MIP 1129)

In a round bottom flask 4-bromo-phenyl isocyanate (100 mg, 0.50 mmol)was dissolved in DCM (10 mL).2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester (246 mg, 0.50 mmol) was added and stirred for 3 hours. Thereaction mixture was evaporated to dryness and the crude mixture waspurified via flash column chromatography 2:1 hexanes/ethyl acetate toafford the tert-butyl ester as a white solid which was deprotected usingTFA/CH₂Cl₂ affording the desired product

¹H NMR (400 MHz, DMSO-d₆) δ 12.5 (s, 3H), 8.55 (s, 1H), 7.35 (d, 4H),6.30 (t, 2H), 6.18 (t, 1H), 4.08 (m, 2H), 3.05 (m, 2H), 2.22 (m, 2H),1.90 (m, 1H), 1.68 (m, 2H), 1.52 (m, 1H), 1.40 (m, 2H), 1.30 (m, 2H).ESMS m/z: 518 (M+H)⁺.

2-(3-{1-Carboxy-5-[3-(4-chloro-phenyl)-ureido]-pentyl}-ureido)-pentanedioicacid (13) (MIP 1110)

In a round bottom flask 4-chloro-phenyl isocyanate (100 mg, 0.65 mmol)was dissolved in DCM (10 mL)2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid, di-t-butylester (318 mg, 0.65 mmol) was added and stirred for 3 hours. Thereaction mixture was evaporated to dryness and the crude mixture waspurified via flash column chromatography 2:1 hexanes/ethyl acetate toafford the tert-butyl ester as a white solid (470 mg, 96%) which wasdeprotected using TFA/CH₂Cl₂ affording the desired product

¹H NMR (400 MHz, DMSO-d₆) δ 12.5 (s, 3H), 8.35 (s, 1H), 7.40 (dd, 2H),7.19 (dd, 2H), 6.30 (t, 2H), 6.10 (t, 1H), 4.08 (m, 21H), 3.05 (m, 2H),2.32 (m, 2H), 1.90 (m, 1H), 1.68 (m, 2H), 1.52 (m, 1H), 1.40 (m, 2H),1.30 (m, 2H). ESMS m/z: 474 (M+H)⁺.

(S)-2-(3-((R)-1-carboxy-5-(□yridine□ne-1-ylmethylamino)pentyl)ureido)pentanedioicacid. (14) (MIP-1108)

The same general procedure as shown in Scheme A, using previouslyprepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester. The compound was deprotected using the previously describedmethods to yield the desired product (51 mg, 70%) as an off-white solid.¹H NMR (400 MHz, DMSO-d₆) δ 8.9 (br, 3H), 7.95 (m, 5H), 7.6 (m, 2H),6.35 (br, 2H), 4.1 (m, 4H), 2.9 (br, 2H), 2.55 (m, 2H), 2.25 (m, 2H),1.70 (m, 4H), 1.3 (m, 2H). ESMS m/z: 460 (M+H)⁺.

2-(3-{-Carboxy-5-[3-(3-iodo-benzyl)-ureido]-pentyl}-ureido)-pentanedioicacid (15) (MIP-1101)

The same general procedure as shown in Scheme 2, using previouslyprepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester. The compound was deprotected using the previously describedmethods to yield the desired product. ESMS m/z: 579 (M+H)⁺.

(19S,23S)-2-(4-iodobenzyl)-1-(4-iodophenyl)-13,21-dioxo-2,14,20,22-tetraazapentacosane-19,23,25-tricarboxylicacid (16) (MIP-1130)

The same general procedure as shown in Scheme A, using previouslyprepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester. The compound was deprotected using the previously describedmethods to yield the desired product (8.3 mg, 10%) as an off-whitesolid. ¹H NMR (400 MHz, DMSO-d₆) δ 7.8 (d), 7.3 (d), 6.3 (dd), 4.25(br), 4.05 (m), 2.97 (m), 2.85 (br), 2.22 (m), 2.05 (m), 0.90 (m), 1.64(m), 1.48 (m), 1.35 (m), 1.2 (m). ESMS m/z: 936 (M+H)⁺.

Rhenium General Experimental:

The rhenium complexes of the SAAC-inhibitors are conveniently isolatedfrom the reactions of the readily available precursor[Net₄]₂[Re(CO)₃Br₃] with the SAAC-inhibitor. Since the donor setsprovided by the SAAC terminus are well documented as effective chelatorsfor the {M(CO)₃}⁺¹ core and have been designed to adopt the requiredfacial arrangement about the metal site, the preparations of thecomplexes were unexceptional.

The {Re(I)(CO)₃}⁺ system followed similar reaction chemistry to that ofthe Tc-99m tricarbonyl core. The use of [Net₄]₂[ReBr₃(CO)₃], as thestarting material led to facile formation of the fac-{Re(CO)₃(L)₃}core.The [Net₄]₂[ReBr₃(CO)₃] was readily derived from the [ReBr(CO)₅]. Thesynthesis of the Re(I) complexes was accomplished by reacting[Net₄]₂[ReBr₃(CO)₃] with the appropriate TEC ligand in the ratio of1:1.2 in 10 ml of methanol. The reaction was allowed to heat at 80° C.for 4 hours. After cooling all of the following reaction products wereall purified using a small silica column with yields ranging from10-30%.

Glu-urea-Lys-PEG2-ReDP:

[Re(CO)₃{(17R,21S)-11,19-dioxo-1-(□yridine-2-yl)-2-(□yridine-2-ylmethyl)-5,8-dioxa-2,12,18,20-tetraazatricosane-17,21,23-tricarboxylicacid}][Br]. (17) (MIP-1133)

The PEG2 dipyridyl compound,(17R,21S)-11,19-dioxo-1-(□yridine-2-yl)-2-(□yridine-2-ylmethyl)-5,8-dioxa-2,12,18,20-tetraazatricosane-17,21,23-tricarboxylicacid was prepared employing the same general procedure as shown inScheme 1, using previously prepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester. The rhenium ester complex was prepared employing the sameprocedure as described in the general rhenium experimental. The compoundwas deprotected using the previously described methods to yield thedesired product (2 mg, 20%) as an off-white solid. ¹H NMR (400 MHz,DMSO-d₆) δ 8.8 (d), 8.00 (dd), 7.55 (d), 7.42 (dd), 6.45 (s), 3.95 (m),3.4-3.6 (m), 2.45 (m), 1.25 (m), 1.1 (m), 0.8 (m). ESMS m/z: 931 (M+H)⁺.

Glu-urea-Lys-PEG4-ReDP:

[Re(CO)₃{(23R,27S)-17,25-dioxo-1-(pyridin-2-yl)-2-(pyridin-2-ylmethyl)-5,8,11,14-tetraoxa-2,18,24,26-tetraazanonacosane-23,27,29-tricarboxylicacid}][Br]. (18) (KM 1-200)

The PEG4 dipyridyl compound(23R,27S)-17,25-dioxo-1-(pyridin-2-yl)-2-(pyridin-2-ylmethyl)-5,8,11,14-tetraoxa-2,18,24,26-tetraazanonacosane-23,27,29-tricarboxylicacid was prepared employing the same general procedure as shown inScheme A, using previously prepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester. The rhenium ester complex was prepared employing the sameprocedure as described in the general rhenium experimental. The compoundwas deprotected using the previously described methods to yield thedesired product. (5.1 mg, 29.6%) as a white solid. ESMS m/z: 1019(M+H)⁺.

Glu-urea-Lys-PEG8-ReDP:

[Re(CO)₃{(35R,39S)-29,37-dioxo-1-(□yridine-2-yl)-2-(□yridine-2-ylmethyl)-5,8,11,14,17,20,23,26-octaoxa-2,30,36,38-tetraazahentetracontane-35,39,41-tricarboxylicacid}][Br]. (19) (MIP-1132)

The PEG8 dipyridyl compound,(35R,39S)-29,37-dioxo-1-(pyridin-2-yl)-2-(pyridin-2-ylmethyl)-5,8,11,14,17,20,23,26-octaoxa-2,30,36,38-tetraazahentetracontane-35,39,41-tricarboxylicacid was prepared employing the same general procedure as shown inScheme A, using previously prepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester. The rhenium ester complex was prepared employing the sameprocedure as described in the general rhenium experimental. The compoundwas deprotected using the previously described methods to yield thedesired product (8.0 mg, 30.4%) as a white solid. ESMS m/z: 1195 (M+H)⁺.

Glu-urea-Lys-C11PAMA-Re:

[Re(CO)₃{(19R,23S)-13,21-dioxo-2-(□yridine-2-ylmethyl)-2,14,20,22-tetraazapentacosane-1,19,23,25-tetracarboxylicacid}](20) (MIP-1109)

The C11-PAMA compound,(19R,23S)-13,21-dioxo-2-(□yridine-2-ylmethyl)-2,14,20,22-tetraazapentacosane-1,19,23,25-tetracarboxylicacid was prepared employing the same general procedure as shown inScheme A, using previously prepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester. The rhenium ester complex was prepared employing the sameprocedure as described in the general rhenium experimental. The compoundwas deprotected using the previously described methods to yield thedesired product (3.0 mg, 75%) as an off-white solid. ESMS m/z: 922(M+H)⁺.

Table 1 below is a summary of synthesized PSMA inhibitors investigated.

TABLE 1 Table 1. Summary of in vitro cell binding data of the additionalor retested Glu-Urea-Lys derivatives. Compound MIP # X Description IC₅₀(nM) — — PMPA  10 1033 — Glu-urea-Lys 498 1137 2-Cl 2-Cl-benzyl 245 11313-Cl 3-Cl-benzyl 277 1135 4-Cl 4-Cl-benzyl  2 1106 H Des-halo benzyl2960  1111 H Des-halo diurea  12 1129 4-Br 4-Br-diurea  2 1110 4-Cl4-Cl-diurea  4 1108 — 2-naphyl 154 1101 3-I 3-I-diurea  10 1130 4-di-IC11 4-di-iodo 300 1133 — PEG2Re 227 KM11-200 — PEG4Re NA 1132 — PEG8Re1747  1109 — C11PAMA-Re 696 1027 4-I 4-I-benzoyl   3* 1095 4-I4-I-diurea  10*β-Amino Acid Analogs

β-amino acid analogs of MIP-1072, MIP-1095, MIP-1027 specifically butthe extension to other analogs such as the technetium conjugates as wellas other halogen analogs is very desirable. We have no new examples tosupport this claim at this time.

Synthesis of {Re(CO)₃}⁺¹ Core Model Complexes

The properties of the Group VII metals technetium and rhenium are verysimilar due to their periodic relationship. It was anticipated that themetals would demonstrate similar reaction chemistry, which is often thecase for the tricarbonyl, nitrogen, and, thiazole chemistry of these twometals. Likewise, due to their similar size that stabilizes the spinpaired d⁶ electron configuration of M(I), perrhenate and pertechnetatehave very similar reaction behaviors. Synthesizing the rhenium-TECsallowed us a facile route to structurally characterize the products. Theperiodic relationship between Tc and Re indicates that Tc-99mradiopharmaceuticals can be designed by modeling analogous rheniumcomplexes.

Some of the new compounds were synthesized with macroscopic quantitiesof rhenium for characterization by conventional methods, includingmass-spectrometry, ¹H and ¹³C NMR spectrometry. Following purification,the synthesized rhenium complexes were run through a HPLC column forpurification and identification of retention times to compare with. Tcreaction products. The rhenium-TEC complexes were also crystallized.

The rhenium complexes of the SAAC-inhibitors are conveniently isolatedfrom the reactions of the readily available precursors {Re(CO)₃(H₂O)₃}⁺¹and [Net₄]₂[Re(CO)₃Br₃] with the SAAC-inhibitor. Since the donor setsprovided by the SAAC terminus are well documented as effective chelatorsfor the {M(CO)₃}+⁺¹ core and have been designed to adopt the requiredfacial arrangement about the metal site, the preparations of thecomplexes were unexceptional.

General Experimental

The {Re(I)(CO)₃}⁺ system followed similar reaction chemistry to that ofthe Tc-99m tricarbonyl core. The use of [Net₄]₂[ReBr₃(CO)₃], as thestarting material led to facile formation, of the fac-{Re(CO)₃(L)₃}core.The [Net₄]₂[ReBr₃(CO)₃] was readily derived from the [ReBr(CO)₅]. Thesynthesis of the Re(I) complexes was accomplished by reacting[Net₄]₂[ReBr₃(CO)₃] with the appropriate TEC ligand in the ratio of1:1.2 in 10 ml of methanol. The reaction was allowed to heat at 80° C.for 4 hours. After cooling all of the following reaction products wereall purified using a small silica column with yields ranging from10-30%.

[Re(CO)₃(2-{3-[3-(Bis-pyridin-2-ylmethyl-amino)-1-carboxy-propyl]-ureido}-pentanediethylester)][Br] (24)

¹H NMR (400 MHz, DMSO-d₆) δ 8.65 (dd, 2H), 7.85 (dd, 2H), 7.7 (dd, 4H),7.25 (dd, 2H), 6.42 (dd, 1H), 6.0 (dd, 1H), 4.5 (m, 2H), 4.16 (m, 2H),3.80 (m, 4H), 2.45 (m, 2H), 2.0 (dd, 2H), 1.5 (m, 4H), 1.25 (m, 6H).ESMS m/z: 812-815.

[Re(CO)₃(2-{3-[5-(Bis-pyridin-2-ylmethyl-amino)-1-carboxy-pentyl]-ureido}-pentanedioicacid)][Br](25) (MIP 1029)

¹H NMR (400 MHz, DMSO-d₆) δ 12.6 (s, 2H), 8.91 (s, 1H), 8.63 (dd, 2H),7.85 (dd, 2H), 7.75 (dd, 4H), 7.3 (dd, 2H), 6.44 (d, 1H), 6.28 (d, 1H),4.45 (s, 2H), 4.10 (m, 2H), 3.15 (s, 1H), 2.60 (m, 2H), 2.25 (m, 2H),1.90 (m, 1H), 1.78 (m, 2H), 1.45 (m, 2H). ESMS m/z: 770-774.

2-{3-[1-Carboxy-5-(carboxymethyl-pyridin-2-ylmethyl-amino)-pentyl]-ureido}-pentanedioicacid (26)

The same general procedure, using the previously prepared and protected2-[3-(5-Amino-1-carboxy-pentyl)-ureido]-pentanedioic acid di t-butylester. The compound was deprotected using the previously describedmethods (2.2 mg, 65%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.65 (d, 1H), 7.91(dd, 1H), 7.56 (d, 1H), 7.45 (dd, 1H), 6.31 (m, 2H), 4.34 (s, 2H), 4.08(m, 4H), 3.10 (m, 2H), 2.24 (m, 2H), 1.95 (m, 1H), 1.68 (m, 4H), 1.5 (m,1H), 1.22 (m, 2H). ESMS m/z: 469 (M+H)⁺. M+1 469.

[Re(CO)₃(2-{3-[1-Carboxy-5-(carboxymethyl-pyridin-2-ylmethyl-amino)-pentyl]-ureido}-pentanedioic acid)](27)

¹H NMR (400 MHz, DMSO-d₆) δ 8.75 (d, 1H), 8.13 (dd, 1H), 7.69 (d, 1H),7.57 (dd, 1H), 6.45 (m, 2H), 4.75 (m, 1H), 4.50 (m, 1H), 4.20 (m, 2H),3.61 (m, 4H), 3.15 (m, 2H), 2.38 (m, 1H), 2.0 (m, 2H), 1.75 (m, 4H),1.62 (m, 1H), 1.25 (m, 2H). ESMS m/z 779-782 (M+2Na)⁺.

Synthesis of Glu-Urea-Lvs(N-benzyl-X) analogs (3)

The compounds of the general structure 3 were prepared in overall yieldsranging from 20-40% using the general route depicted in Scheme A. Thekey synthetic intermediate (1) was reacted with the appropriate aldehydeat room temperature in for one hour to form the □yridi baseintermediate. The □yridi base was not isolated but was reduced in situwith sodium triacetoxyborohydride. The t-butyl ester protecting groupswere removed using 50% TFA in DCM for 1 hour at room temperature. Uponcompletion of the deprotection, the reactions were concentrated on arotary evaporator and purified by HPLC or flash chromatography to affordthe desired products (3) in 40-80% yield.

Synthesis of Glu-Urea-Ureido(Phenyl-X) Analogs

The following compounds of the general structure 8 were prepared inoverall yields ranging from 20-60% by the route depicted in Scheme B:The key synthetic intermediate (4) was reacted with the appropriatephenylisocyanate at room temperature to afford the desired protectedintermediates (5) in good yields. The t-butyl ester protecting groupswere removed ii the presence of 50% TFA in DCM for 1 hour at roomtemperature. Upon completion, the reactions were concentrated on arotary evaporator purified by HPLC or recrystallization to afford thedesired products (6) in 40-90% yield.

Preparation and Characterization of the Radio-Labeled ComplexesTechnetium-99m Labeling

Preparation of the ^(99m)Tc-labeled complexes were achieved by additionof 100 μL of a solution containing [^(99m)Tc(CO)₃(H₂O)₃]⁺ to 500 μL of10⁻⁴M solutions of the inhibitor-SAAC. The mixtures were heated at 70°C. for 30 min. The products were analyzed for their radiochemical purityby reverse-phase HPLC.

The stability of the radiolabeled compounds in solution and in serumwere determined as a function of time and solution conditions.Specifically, after radiolabeling and isolation, the product was storedat room temperature for 6 h after which HPLC analysis was performed tocheck for degree of label retention, as well as potential productdegradation. The reformation of TcO₄ ⁻ and the presence of the reducedmaterial TcO₂ was analyzed.

To assist in predicting the in vivo stability, ligand challenges wereperformed. Specifically, the stabilities of the ^(99m)Tc complexes wereinvestigated by incubating the HPLC purified complexes in 5% mouse serumat room temperature and 37° C. The ability of competing ligands, such ascysteine and DTPA, to extract Tc-99m from the complexes was studied byincubating the purified complexes with solutions (PBS pH 7.2) containingcompeting ligands at final concentrations of 0.1 M.

The results of the labeling competition studies demonstrated nodegradation of the Tc-99m-complexes out to 6 hours in the serum or thecompeting ligands study. The results of the incubation at 37° C. after 6hours are shown in FIG. 2.

Iodinations of DCT

Preparation of the iodine-131 labeled compoundN—[N—[(S)-1,3-dicarboxypropyl]carbamoyl]-S-3-iodo-L-tyrosine(I-131-DCIT) was achieved by addition of 100 ul of [I-131] NaI in 0.1 NNaOH to a PBS (pH 7.2) solution containing. DCT (1 mg/mL) in an IodogenTube™ (Fisher Scientific, Pierce). The mixture was vortexed for 3minutes and stored at room temperature for 20 minutes.

The stability of the radiolabeled compound in solution was determined asa function of time. Specifically, after radiolabeling and isolation, theproduct was stored at room temperature for 48 h after which HPLCanalysis was performed to check for degree of label retention, as wellas potential product degradation. The reformation of NaI and thepresence of the reduced iodates was analyzed. The results of thelabeling stability study demonstrated no significant degradation of theI-131 DCIT out to 2 days at room temperature. The results of the studyare shown in FIG. 3.

Preparation of the iodine-131 labeled compound2-{3-[1-Carboxy-5-(4-iodo-benzoylamino)-pentyl]-ureido}-pentanedioicacid (I-131-MIP 1072) was achieved by addition of 100 ul of [I-131] NaIin 0.1 N NaOH with 30 μl methanol with 0.5% acetic acid to a PBS (pH7.2) solution containing MIP 1072 (1 mg/mL) in an IODOGEN TUBE (FisherScientific). The mixture was vortexed for 3 minutes and stored at roomtemperature for 20 minutes.

The stability of the radiolabeled compound in solution was determined asa function of time. Specifically, after radiolabeling and isolation, theproduct was stored at 37° C. for 3 days after which HPLC analysis wasperformed to check for degree of label retention, as well as potentialproduct degradation. The reformation of NaI and the presence of thereduced iodates was analyzed. The results of the labeling stabilitystudy demonstrated no significant degradation of the I-131 1072 out to 3days at room temperature in DMSO, 10% ethanol/saline, PBS pH 7.2, and 6%ascorbate/3% gentisic acid solution. The results of the study are shownin FIG. 4.

Biological Characterization of SAAC-Urea-Glutamate Conjugates

The newly prepared SAAC-urea-Glu conjugates were screened in a humanprostate cancer cell binding assay using PSMA-positive, LnCap cells, andPSMA-negative, PC3 cells. Compounds demonstrating specific uptake orbinding to PSMA-positive cells will be studied for tumor localization invivo.

In Vitro Cold Screening Assays Verses I-131 DCIT. LNCaP and PC3 humanprostate cancer cells were obtained from American Type CultureCollection, Rockville, Md. LNCaP cells were maintained in RPMI-1640medium supplemented with 10% fetal bovine serum (FBS). PC3 cells weregrown in F12K medium supplemented with 10% FBS. Binding of theradiolabeled compound and competition with cold derivatives to LNCaP andPC-3 cells was performed according to the methods of Tang et al. (Tang,H.; Brown, M.; Ye, Y.; Huang, G.; Zhang, Y.; Wang, Y.; Zhai, H.; Chen,X.; Shen, T. Y.; Tenniswood, M., Prostate targeting ligands based onN-acetylated alpha-linked acidic dipeptidase, Biochem. Biophys. Res.Commun. 2003, 307, 8-14) with appropriate modifications. Cells wereplated in 12-well plates at approximately 4×10⁵ cells/well and incubatedfor 48 hours in a humidified incubator at 37° C./5% carbon dioxide priorto addition of compound. Each unique SAAC-urea-Glu conjugate wasprepared and diluted in serum-free cell culture medium containing 0.5%bovine serum albumin (BSA) in combination with 3 nM I-131 DCIT (knowninhibitor). Total binding was determined by incubating I-131 DCITwithout test compound. Plates were incubated at room temperature for 1hour. Cells were removed from the plates by gently pipeting andtransferred to eppendorff tubes. Samples were microcentrifuged for 15seconds at 10K×g. The medium was aspirated and the pellet was washedtwice by dispersal in fresh assay medium followed bymicrocentrifugation. Cell binding of I-131 DCIT was determined bycounting the cell pellet in an automated gamma counter. Nonspecificbinding was determined as the counts associated with the cells afterincubating with 2 uM nonradiolabeled compound or2-phosphonomethyl-pentanedioic acid (PMPA). The control compounds aredepicted below.

The two key compounds for the binding assays, are shown above: theI-DCIT (Kozikowski et al) and 2-Phosphonomethyl-pentanedioic acid(PMPA—right), a potent inhibitor with IC₅₀=6 nM.

(ii) In Vitro Dose Screening. I-131 DCIT bound specifically to LnCapcells and not PC3 cells as is evident by the counts displaceable bynonradiolabeled compound or PMPA in LnCap cells only (FIG. 5). Bindingconstants were determined by incubating LnCap cells with various amountsof nonradiolabeled DCIT in the presence of a constant amount of I-131DCIT and dividing by the specific activity of each solution to determinethe number of fmoles compound bound (FIG. 6). The Kd was determined tobe 264 nM and Bmax was 254 fmoles. Compounds MIP-1008 and MIP-1033 whichat 2 uM competed with I-131 DCIT for binding to LnCap cells, wereretested at various doses to determine IC-50 values (FIGS. 7 and 8).While MIP-1072, MIP-1095, and MIP-1097 displayed IC50 values<50 nmcompounds MIP-1008 and MIP-1033 exhibited IC-50s of 98 nM and 497 nM,respectively. Compounds MIP-1025, MIP-1028, and MIP-1029 did not competefor binding (Table 1).

In order to confirm the results of the Scatchard analysis of FIG. 7indicating MIP-1072 internalization into LNCaP cells, the rate of uptakeof MIP-1072 in LNCaP cells was monitored. Each well was dosed with 100nM MIP-1072 (2 uCi/well) at 4° C. and 37° C. Binding to PSMA reachedequilibrium after 15 min as evidenced by the plateau in the curve at 4°C. The cells incubated at 37° C. continued to internalize MIP-1072 afterequilibrium had been reached. This result. FIG. 10, confirms theScatchard and indicates that MIP-1072 is indeed internalized.

(iii) Microsome Assay Experimental

Pooled male rat liver microsomes (1 mg/mL, BD Biosciences), NADPHregenerating system (1.3 mM NADP, 3.3 mM glucose 6-phosphate and 0.4U/mL glucose 6-phosphate dehydrogenase, BD Biosciences) and testcompound (50 μM MIP-1072, 50 μM DCT, and 100 μM phenacetin) were addedto 0.1 M potassium phosphate buffer (pH 7.4) in order to monitor thecatastrophic degradation of the test compounds. The mixture wasincubated at 37° C. and at the indicated time (0, 15, 60 min) thereaction was stopped by the addition of an equal volume of ice coldmethanol (500 μL). The resulting slurry was then centrifuged at 21,000×Gfor 10 min and the supernatant was collected and injected onto anAgilent LCMS model MSD SL using a 95:5 water:acetonitrile (with 0.1%formic acid) to 40:60 water:acetonitrile (with 0.1% formic acid)gradient and monitoring for the parent ion only in single ion mode. Theresults, shown in FIGS. 11A and 11B, are expressed as degradation of theparent ion with respect to the 0 min time point.

The stability of MIP-1072 was assessed using rat liver microsomes.MIP-1072 (50 μM) and Phenacetin (100 μM) were incubated with rat livermicrosomes at 37° C. for the indicated time. Phenacetin was used as acontrol substance that is known to be metabolized, MIP-1072 was notdegraded by the rat liver microsomes during the incubation period.However, phenacetin was degraded by 22% after a 60 min incubation.

The lead compound, MIP 1072, was I-131-labeled for tissue distributionstudies in mice with both LNCaP (PSMA positive) and PC3 (PSMA negative)tumors implanted. The compound was radiolabeled by the route shownbelow:

The tissue biodistribution results, were consistent with the in-vitrodata, and demonstrated significant uptake in the LNCaP (PSMA positive)tumors. The results also displayed a high degree of specificity withvery little activity in the PC3 (PSMA negative) tumors. A graphdepicting the mice distribution is shown below (FIG. 12).

The biological assessment usingN—[N—[(S)-1,3-dicarboxypropyl]carbamoyl]-S-3-iodo-L-tyrosine(I-131-DCIT) verses “cold” complexes proved to be a rapid first screen,followed by dose curves to determine accurate IC₅₀ values. The leadseries of compounds that exhibited IC50 values<50 nM. In vivo data ofthe lead series demonstrated high affinity, with 3% ID/g accumulating inthe LNCaP tumors, and high specificity with the LNCaP-to-PC3 ratioexceeding 15-to 1.

LNCaP Cell Lysis Protocol

-   2 confluent T75 Flasks.-   Wash cells off the plate by pipetting up and down with media.-   Wash with 0.32 M sucrose, re-centrifuge-   Re-suspend cell pellet in 1 mL 50 mM Tris-HCl, pH 7.4, 0.5% Triton    X-100-   Centrifuge at 14000 rpm for 1 min to precipitate nuclei-   Remove supernatant and divide into 50 uL aliquots-   Store at −80 C    Protein Assay:-   Bio-Rad Protein Standard II—1.44 mg/ml-   Since using detergent in lysis step, make working reagent, A′ by    adding 20 uL of reagent S to each 1 mL of reagent A that will be    needed for the run. (If a precipitate forms, warm and vortex)-   Prepare 5 protein dilutions—0, 0.2, 0.4, 0.8, 1.6 mg/mL-   Also prepare 1/10, 1/100, and 1/1000 dilutions of the unknown-   Combine 25 μL standard/unknown, 100 μL A′, 800 μL reagent B in    duplicate. Mix-   After ˜15 min measure absorbance at 750 nM    NAALADase Assay:-   Rxn Buffer: 50 mM Tris-HCl, pH 7.4, 20 mM CoCl2, 32 mM NaCl-   Make cold NAAG (100 mM stock) dilute 1/100 in Rxn Buffer for 1 mM-   Combine 600 uL buffer and LNCaP cell lysate (200 μg) Pre-incubate 37    C for 3 min-   Pre-incubate Rxn Buffer and LNCaP cell lysate for 3 min at 37 C-   Add 6 μL of 1 mM NAAG (for 1 μM final cone) spiked with 1,000,000    CPM of ³H-NAAG (100 μL of 1 mM NAAG+10 μL of 3H-NAAG (10 μCi)). For    competition add PMPA.-   Incubate for 30 min-   At indicated time, stop reaction by removing 100 uL of the    reaction-mix and adding an equal volume of ice cold 0.25 M KH₂PO₄,    pH 4.3 to stop the rxn.-   Apply ½ of mixture to 250 mg AG 50W-X4 cation exchange column    (200-400 mesh, H⁺ form, swell resin with D1 H2O prior to use), Save    the other ½ for counting.-   Wash column with 500 μL 1:1 Rxn Buffer/0.25MKH₂PO₄-   Elute with 3M KCl (1.5 mL)-   Count 100 uL of the load, elution and reaction (diluted 1:6) to    minimize quenching

Notes:

-   Time=0 control values will be subtracted from experimental time    points-   Results expressed as pmol ³H-glutamate formed/min/mg protein-   Grant says inc only 10 min to ensure linearity, although    Luthi-Carter, et al (J Pharm Exp Therap 1998 286(2)) says 2 hours    still no effect on linearity and less than 20% of the substrate    consumed    Therapeutic Treatments

Compounds of the present can be used to inhibit NAALADase fortherapeutic treatments. Diseases that could be receptive to NAALADasetreatment include painful and sensory diabetic neuropathy, neuronaldamage and prostate cancer, schizophrenia, colorectal cancer,inflammation, amyotrophic lateral schlerosis, or diabetic neuropathy.The present compounds can also be used an analgesic. Guidance for themodeling of such therapeutic treatments can be found in Goodman &Gilman's The Pharmacological Basis of Therapeutics, McGraw Hill, 10edition, 2001, Pharmaceutical. Preformulation and Formulation: APractical Guide from Candidate Drug Selection to Commercial Dosage Form,CRC, 2001 and Handbook of Pharmaceutical Excipients. AphA Publications,5 edition, 2005.

Competitive Binding of Analogs (FIG. 16)

The ability of non-radioactive analogs to compete with ¹³¹I-DCIT forbinding to PSMA was tested in the PSMA positive human prostate cancercell line, LNCaP cells. LNCaP cells (300,000 cells/well) were incubatedfor 1 hour with 3 nM [¹³¹I]-DCIT in the presence of 1-10,000 nM MIP-1072in RPMI-1640 medium supplemented with 0.5% bovine serum albumin, thenwashed and counted in a gamma counter. All documents cited in thisspecification including patent applications are incorporated byreference in their entirety.

Direct Binding and Internalization of MIP-1072

The direct binding of ¹²³I-MIP-1072 to prostate cancer cell was examined(FIG. 17). LNCaP cells, or the PSMA negative cell line, PC3 cells, wereincubated in RPMI-1640 medium supplemented with 0.5% bovine serumalbumin for 1 hour with 3 nM ¹²³I-MIP-1072 alone, or in the presence of10 μM unlabeled MIP-1072, or 10 μM 2-(phosphonomethyl)-pentanedioic acid(PMPA), a structurally unrelated NAALADase inhibitor. Cells were washedand counted in a gamma counter.

The affinity constant (K_(d)) of MIP-1072 was determined by saturationbinding analysis (FIG. 18). LNCaP cells were incubated for 1 hour with30-100,000 pM ¹³¹I-MIP-1072 in HBS (50 mM Hepes, pH 7.5, 0.9% sodiumchloride) at either 4° C. or 37° C. in the absence or presence of 10 μMunlabeled MIP-1072 (to determine nonspecific binding). Cells were thenwashed and the amount of radioactivity was measured on a gamma counter.Specific binding was calculated as the difference between total bindingand nonspecific binding. The affinity constant (K_(d)) of theinteraction of MIP-1072 with PSMA on LNCaP cells was determined bysaturation binding analysis performed by titrating ¹²³I-MIP-1072 (3pM-1,000 nM) in the presence and absence of an excess ofnon-radiolabeled MIP-1072 (10 μM). A K_(d) of 4.8 nM, and Bmax of 1,490fmoles/10⁶ cells at 4° C. was determined by nonlinear regressionanalysis using Graph Pad Prism software (FIG. 18). The K_(d) was notsignificantly different at 37° C., 8.1 nM. The Bmax, however, wasgreater at 37° C. than at 4° C.; 1,490 vs. 4,400 fmol/10⁶ cells,respectively, indicating internalization of MIP-1072. The results beloware representative of two independent analyses.

The ability of MIP-1072 to internalize in LNCaP cells was confirmed byan acid wash assay (FIG. 19). LNCaP cells were incubated in HBS with 100nM ¹²³I-MIP-1072 for 0-2 hours at 4 and 37° C. At the indicated time themedia was removed and the cells were incubated in mild acid buffer (50mM glycine, 150 mM NaCl, pH 3.0) at 4° C. for 5 minutes. After the briefincubation the cells were centrifuged at 20,000×g for 5 minutes. Thesupernatant and cell pellet were counted in a gamma counter. In order toconfirm the results of the saturation binding analysis indicatingMIP-1072 internalization into LNCaP cells, we monitored the rate ofuptake of MIP-1072 in LNCaP cells. Each well was dosed with 100 nMMIP-1072 (2 uCi/well) at 4° C. and 37° C. Binding to PSMA reachedequilibrium after 15 min as evidenced by the plateau in the curve at 4°C. The cells incubated at 37° C. continued to internalize MIP-1072 afterequilibrium had been reached. The results show a time dependent, acidinsensitive increase in radioactivity associated with the pellet at 37°C. but not at 4° C., indicating that ¹²³I-MIP-1072 is internalized at37° C. but not at 4° C. (FIG. 19).

Tumor Uptake and Tissue Distribution of ¹²³I-MIP-1072

A quantitative analysis of the tissue distribution of ¹²³I-MIP-1072 wasperformed in separate groups of male NCr Nude^(−/−) mice bearing PSMApositive LNCaP xenografts (approximately 100-200 mm³) administered viathe tail vein as a bolus injection (approximately 2 μCi/mouse) in aconstant volume of 0.05 ml. The animals (n=5/time point) were euthanizedby asphyxiation with carbon dioxide at 0.25, 1, 2, 4, 8, and 24 hourspost injection. Tissues (blood, heart, lungs, liver, spleen, kidneys,adrenals, stomach, large and small intestines (with contents), testes,skeletal muscle, bone, brain, adipose, and tumor) were dissected,excised, weighed wet, transferred to plastic tubes and counted in anautomated γ-counter (LKB Model 1282, Wallac Oy, Finland). To compareuptake of ¹²³I-MIP-1072 in LNCaP versus PC3 tumors, and to demonstratethat the compound was on mechanism via competition with2-(phosphonomethyl)-pentanedioic acid (PMPA), some mice bearing eitherLNCaP or PC3 xenografts were pretreated with 50 mg/kg PMPA 5 minutesprior to injection with ¹²³I-MIP-1072 and selected tissues wereharvested at 1 hour post injection. MIP-1072, uptake and exposure wasgreatest in the kidney and LNCaP xenograft which express high levels ofPSMA. Peak uptake in the kidney was 158±46% ID/g at 2 hours and theLNCaP xenograft was 17±6% ID/g at 1 hours (FIG. 20). Uptake in thesetarget tissues was rapid, whereas the washout was slower in the LNCaPxenograft. ¹²³I-MIP-1072 was demonstrated to be on mechanism in vivo asevidenced by the localization to PSMA expressing LNCaP tumors but notPC3 tumors which do not express PSMA (FIG. 21). In addition, both thetumor and kidneys were blocked by pretreating the mice with PMPA, apotent inhibitor of PSMA.

What is claimed is:
 1. A method of treating a patient with prostatecancer, the method comprising a step of: administering a therapeuticallyeffective amount of a glutamate-urea-lysine prostate-specific membraneantigen (PSMA)-binding moiety, wherein the glutamate-urea-lysinePSMA-binding moiety comprises a structure:

or a pharmaceutically acceptable salt thereof, wherein each Z,independently, is H or C₁-C₄ alkyl.
 2. A method of treating a patientwith prostate cancer, the method comprising a step of administering atherapeutically effective amount of a glutamate-urea-lysineprostate-specific membrane antigen (PSMA)-binding moiety, wherein theglutamate-urea-lysine PSMA-binding moiety corresponds to a compound ofFormula (I):

wherein R is a C₆-C₁₂ substituted or unsubstituted aryl, a C₃-C₁₂substituted or unsubstituted heteroaryl, a C₁-C₆ substituted orunsubstituted alkyl or —NR′R′; Q is C(O), O, NR′, S(O)₂, C(O)₂, or(CH₂)_(p); Y is C(O), O, NR′, S(O)₂, C(O)₂, or (CH₂)_(p); each Z,independently, is H or C₁-C₄ alkyl; m is 0, 1, 2, 3, 4 or 5; n is 0, 1,2, 3, 4, 5 or 6; p is 0, 1, 2, 3, 4, 5 or 6; and R′ independently is H,a C₆-C₁₂ substituted or unsubstituted aryl, a C₃-C₁₂ substituted orunsubstituted heteroaryl or a C₁-C₆ substituted or unsubstituted alkyl,when substituted, aryl, heteroaryl and alkyl are substituted withhalogen, C₃-C₁₂ heteroaryl, —NR′R′ or COOZ; further wherein at least oneof Q and Y comprises a heteroatom selected from the group consisting ofO, S, and N; and (i) at least one of R or R′ is a C₆-C₁₂ aryl or C₃-C₁₂heteroaryl substituted with a radiohalogen or (ii) at least one of R orR′ is a C₃-C₁₂ heteroaryl; or a pharmaceutically acceptable salt of thecompound of Formula (I).
 3. The method of claim 2, wherein n is 0 or 1;m is 0, 1, 2, 3 or 4; Q is NR′; Y is C(O) or CH₂; and R is a C₆-C₁₂substituted or unsubstituted aryl.
 4. The method of claim 3, wherein Ris a phenyl moiety substituted with a radiohalogen.
 5. The method ofclaim 2, wherein the compound is:

wherein the hydrogen of the carboxy groups of any of the above compoundscan be substituted with C₁-C₄ alkyl, and X is selected from the groupconsisting of radiohalogen I-123, I-125, I-131, I-124, Br-75, Br-77, andF-18.
 6. The method of claim 2, wherein the compound is:


7. The method of claim 2, wherein n is 0 or 1; m is 0, 1, 2, 3 or 4; Qis NR′; Y is C(O) or CH₂; R is a —CH₂(CH₂)₁₋₄CHNR′R′; and R′independently is a C₁-C₂ alkyl substituted with a pyridine or carboxygroup.
 8. A method of treating a patient with prostate cancer, themethod comprising the step of: administering a therapeutically effectiveamount of a compound of formula (III):

wherein L is a bond, an optionally substituted C₁-C₁₅ alkyl, C₁-C₁₅alkenyl, or C₁-C₁₅ alkynyl, or —[(CH₂)_(q)O]_(s)—, wherein q and s areindependently an integer of 1 to 10; E is —NR′R′; Q is C(O), O, NR′, S,S(O)₂, C(O)₂, or (CH₂)_(p); Y is C(O), O, NR′, S, S(O)₂, C(O)₂, or(CH₂)_(p); each Z, independently, is H or C₁-C₄ alkyl; m is 0, 1, 2, 3,4 or 5; n is 0, 1, 2, 3, 4, 5 or 6; n′ is 0, 1, 2, 3, 4, 5 or 6; p is 0,1, 2, 3, 4, 5 or 6; R′ independently is H, a C₆-C₁₂ substituted orunsubstituted aryl, a C₃-C₁₂ substituted or unsubstituted heteroaryl ora C₁-C14 substituted or unsubstituted alkyl, when substituted, aryl,heteroaryl and alkyl are substituted with a radiohalogen, C₃-C₁₂heteroaryl, —NR′R′ or COOZ; or a pharmaceutically acceptable saltthereof.
 9. The method of claim 8, wherein the compound is:

wherein the hydrogen of the carboxy groups of the above compounds can besubstituted with C₁-C₄ alkyl.
 10. The method of claim 2, wherein thecompound is of Formula (Iib):

wherein —Y—R is selected from the group consisting of:

wherein X is H or a radiohalogen selected from the group consisting of:I, Br, Cl, and F.
 11. The method of claim 2, wherein the compound is ofthe following structure:

wherein the hydrogen of the carboxy groups of the compound or salt canbe substituted with C₁-C₄ alkyl; and X is selected from a radiohalogenand H.
 12. The method of claim 2, wherein the compound is of thefollowing structure:

wherein the hydrogen of the carboxy groups of the compound or salt canbe substituted with C₁-C₄ alkyl; and X is selected from a radiohalogenand H.
 13. The method of claim 2, wherein the compound is selected fromthe group consisting of:


14. The method of claim 2 wherein the compound is selected from thegroup consisting of:


15. The method of claim 2, wherein the compound is selected from thegroup consisting of:


16. The method of claim 10, wherein X is a radiohalogen of I, Br, Cl orF.
 17. The method of claim 10, wherein the radiohalogen is a therapeuticradiohalogen.
 18. The method of claim 10, wherein the radiohalogen isI-123, I-125, I-131, I-124, Br-75, Br-77, or F-18.
 19. The method ofclaim 2, wherein the radiohalogen is a therapeutic radiohalogen.
 20. Themethod of claim 2, wherein the radiohalogen is I-123, I-125, I-131,I-124, Br-75, Br-77, or F-18.
 21. The method of claim 8, wherein n′ is0.
 22. The method of claim 21, wherein L is an optionally substitutedC₁-C₁₅ alkyl.
 23. The method of claim 22, wherein n is
 0. 24. The methodof claim 23, wherein Q is NR′.
 25. The method of claim 22, wherein Q isNR′ and Y is C(O).
 26. The method of claim 24, wherein R′ independentlyis H, a C₃-C₁₂ substituted or unsubstituted heteroaryl, or a C₁-C₁₅substituted or unsubstituted alkyl.
 27. The method of claim 25, whereinR′ independently is H, a C₃-C₁₂ substituted or unsubstituted heteroaryl,or a C₁-C₁₅ substituted or unsubstituted alkyl.
 28. The method of claim26, wherein R′ independently is a C₃-C₁₂ substituted or unsubstitutedheteroaryl or a C₁-C₁₅ substituted alkyl, and when substituted, theC₁-C₁₅ substituted alkyl is substituted with COOH.
 29. The method ofclaim 27, wherein R′ independently is a C₃-C₁₂ substituted orunsubstituted heteroaryl or a C₁-C₁₅ substituted alkyl, and whensubstituted, the C₁-C₁₅ substituted alkyl is substituted with COOH. 30.The method of claim 11, wherein X is a radiohalogen.
 31. The method ofclaim 11, wherein X is selected from the group consisting of: I, Br, Cl,F, I-123, I-125, I-131, I-124, Br-75, Br-77, and F-18.
 32. The method ofclaim 11, wherein X is I-123, I-125, I-131, or I-124.
 33. The method ofclaim 11 having the formula:


34. The method of claim 12, wherein X is a radiohalogen.
 35. The methodof claim 12, wherein X is selected from the group consisting of: I, Br,Cl, F, I-123, I-125, I-131, I-124, Br-75, Br-77, and F-18.
 36. Themethod of claim 12, wherein X is I-123, I-125, I-131, or I-124.
 37. Themethod of claim 12 having the formula:


38. The method of claim 2, wherein the compound is of the followingstructure:

wherein the hydrogen of the carboxy groups of the compound or salt canbe substituted with C₁-C₄ alkyl; and X is selected from a radiohalogenand H.
 39. The method of claim 38, wherein X is selected from the groupconsisting of: I, Br, Cl, F, I-123, I-125, I-131, I-124, Br-75, Br-77,and F-18.
 40. The method of claim 38, wherein X is I-123, I-125, I-131,or I-124.
 41. The method of claim 38 wherein the compound has theformula:


42. A method of treating a patient with prostate cancer, the methodcomprising the step of: administering a therapeutically effective amountof a compound having the formula:

wherein X is a radiohalogen selected from the halogen group consistingof: I, Br, Cl, and F; or H; or a pharmaceutically acceptable saltthereof.
 43. The method of claim 42, wherein X is a radiohalogen of I,Br, Cl or F.
 44. The method of claim 42 wherein the compound has theformula:


45. The method of claim 1, wherein the glutamate-urea-lysineprostate-specific membrane antigen (PSMA)-binding moiety provides ananalgesic effect on the patient.
 46. The method of claim 2, wherein theglutamate-urea-lysine prostate-specific membrane antigen (PSMA)-bindingmoiety provides an analgesic effect on the patient.
 47. A method oftreating a patient with prostate cancer, the method comprising a stepof: administering a therapeutically effective amount of aglutamate-urea-lysine prostate-specific membrane antigen (PSMA)-bindingmoiety, wherein the glutamate-urea-lysine PSMA-binding moiety comprisesor is derived from:

or a pharmaceutically acceptable salt thereof.